Poly-.beta.-hydroxybutyrate (pHB) is a biodegradable, biocompatible, thermoplastic made by microorganisms [Baptist, J. N., 1962, U.S. Pat. No. 3,036,959]. In the cell, pHB is an intracellular storage material synthesized and accumulated during unbalanced growth. It accumulates as distinct white granules that are clearly visible in the cytoplasm of the cell. Under conditions of nutrient starvation, pHB is used by the cell as an internal reserve of carbon and energy. Many bacteria including those in the soil, are capable of pHB production and breakdown. Animal cells do not form pHB but are able to break down the polymer.
pHB is a homopolymer of repeating 3-hydroxybutyric acid units. Copolymers with hydroxyvaleric acid can be made by "precursoring" (e.g.) adding propionic acid to the culture during growth [Holmes et al., 1981, European Patent 0,052,459 and 1984 U.S. Pat. No. 4,477,654]. This modification to the pHB homopolymer reduces the crystallinity and melting point of the plastic, allowing film formation and melt-extrusion applications. PHB plastics also are used in microelectronics applications exploiting the piezoelectric properties of pHB. An immediate market for pHB plastics will be in high value added products (e.g.) biodegradable surgical pins, plates, pegs and sutures, implants for drug delivery, and possibly meshes can be used as artificial skin materials. PHB derived plastics also have considerable potential application as biodegradable bulk plastics, replacing non-biodegradable products form from polypropylene or polyethylene. Development of many of these products is ongoing in view of their potential uses.
Production of pHB in the cell occurs during imbalanced growth. Usually this is the stationary phase of bacterial growth, but this can be induced in an actively growing culture by imposing a nutrient (O.sub.2, nitrogen, phosphate, or sulfate) limitation in the presence of excess carbon source. During this imbalance, NADH accumulates and exerts a feedback repression on various enzymes whose activities are essential for the continued growth of the cell. NADH can be oxidized to NAD.sup.+, eliminating this growth inhibition, by the action of acetoacetyl CoA reductase and the polymerization of acetoacetyl CoA into pHB.
NAD.sup.+ is nicotinamide adenine dinucleotide and NADH is its reduced form. NAD.sup.+ is a major electron acceptor in the oxidation of fuel molecules in the cell. NAD.sup.+ fulfills this function by accepting two electrons and two hydrogen ions from substances it oxidizes. Thus, NAD+ becomes NADH. Stryer, Biochemistry 2d ed., W. H. Freeman and Company, San Francisco, pp. 244-246.
Azotobacter vinelandii is a harmless soil microbe that has an obligate O.sub.2 requirement for growth and can use N.sub.2 as a nitrogen source via nitrogen fixation. A. vinelandii normally produces pHB by the methods noted above and much of the early work concerning pHB synthesis was conducted in Azotobacter species. Azotobacter species that produce large amounts of pHB have been reported, but these cells have been unstable and also produce large amounts of capsule and slime, which interfere with pHB extraction and decrease the efficiency of conversion of carbon substrate to pHB.
At present, pHB is produced commercially by ICI in the U.K., using a strain of Alcaligenes eutrophus growing in a glucose salts medium. Their fermentation involves a rapid growth phase (60 h), followed by phosphate-limitation and glucose feeding (an additional 48-60 h). During phosphate-limited growth, pHB is formed and may account for 75% of the total cell weight. The yield per litre is dependent on the initial cell mass and theoretical yields of 0.33 t pHB t.sup.-1 glucose have been calculated [Byrom, D., Trends in Biotechnology 5: 246-250, 1987].
Presently, pHB production involves a long fermentation time, in the stationary phase of growth, to obtain high levels of pHB. Different nutrient limitations have been imposed during stationary phase to enhance pHB production.
Currently, pHB production is limited by a relatively long fermentation time, dependance on amount of pHB produced upon continued cell activity after the active (exponential) phase, dependance on amount of pHB produced upon a pregrowth period to achieve an initial cell mass such that a certain amount of the carbon source is used to produce cell mass rather than pHB, and the need to use relatively expensive substrates (such as glucose) for fermentation.
To date, there appears to have been an effort to increase pHB yield in industrial applications by increasing batch size. Because pHB is considered a secondary metabolite, produced in the stationary phase of growth after active cell growth, the possibility of increasing yield by bringing about pHB production during exponential growth has not been addressed and successfully exploited.
Likewise, the possibility of exploiting relatively unrefined carbon sources in pHB production has not been successfully exploited. The unrefined carbon sources are typically more complex, less refined, or sometimes unpurified materials or even mixtures of materials, which are not necessarily of pure or defined composition. Example of unrefined carbon sources include blackstrap molasses, sugar-beet molasses, carbohydrates and phenols in industrial or municipal wastes. Instead of said unrefined substances, relatively pure carbon sources of defined composition, such as glucose have been used for industrial pHB production to date.